Botulinum neurotoxins are a family of seven bacterial toxins (BoNT/A-G) that cause the disease botulism in humans and animals. They target neurons and cleave host proteins required for synaptic vesicle exocytosis, thereby blocking synaptic transmission and paralyzing the hosts. This mode of action and its role in pathogenesis has been well established. Humans used to get exposed to BoNTs mainly via food poisoning, but significant changes have occurred in human- BoNT interactions. First, medical advances can now revive previously untreatable patients from acute BoNT actions. Second, BoNTs are utilized as therapeutics to target neurons via local injections over long periods of time. These changes have raised the critical need to examine the additional long-term consequences of exposure to BoNTs. Indeed, previous reports and our preliminary studies have revealed that a subset of BoNTs may induce degeneration of cultured rodent neurons in addition to blocking synaptic vesicle exocytosis. In Aim 1, we will examine whether BoNTs may induce degeneration of human neurons and in in vivo rodent models that mimic therapeutic applications. We will also investigate whether human genetic variations might render certain populations susceptible to potential cytotoxicity from the major therapeutic toxin BoNT/A. In Aim 2, we will examine our hypothesis that BoNTs induce neurodegeneration because their substrates play an essential role in maintaining the membrane balance at plasma membranes. Aim 1 will provide a solid foundation for establishing the potential clinical relevance. Aim 2 will provide a mechanistic understanding for BoNT cytotoxicity. Together, these studies will establish neuronal cytotoxicity as a newly emerged long-term consequence due to changes in BoNT-human interactions, with significant implications for understanding long-term effects of botulism in patients and for ensuring the safety of using BoNTs as therapeutic toxins.